Assessment of food sources and the intake of the colourless carotenoids phytoene and phytofluene in Spain

nutrients Article Assessment of Food Sources and the Intake of the Colourless Carotenoids Phytoene and Phytofluene in Spain Begoña Olmedilla-Alonso 1,* , Ana M. Benítez-González 2, Rocío Estévez-Santiago 3, Paula Mapelli-Brahm 2, Carla M. Stinco 2and Antonio J. Meléndez-Martínez 2,*   Citation: Olmedilla-Alonso, B.; Benítez-González, A.M.; Estévez-Santiago, R.; Mapelli-Brahm, P.; Stinco, C.M.; Meléndez-Martínez, A.J. Assessment of Food Sources and the Intake of the Colourless Carotenoids Phytoene and Phytofluene in Spain. Nutrients 2021, 13, 4436. https://doi.org/10.3390/ nu13124436 Academic Editor: Winston Craig Received: 30 October 2021 Accepted: 9 December 2021 Published: 11 December 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Department of Metabolism and Nutrition, Institute of Food Science, Technology and Nutrition (ICTAN-CSIC), C/JoséAntonio Novais, 10, 28040 Madrid, Spain 2Food Colour and Quality Laboratory, Facultad de Farmacia, Universidad de Sevilla, 41012 Sevilla, Spain; [email protected] (A.M.B.-G.); [email protected] (P.M.-B.); [email protected] (C.M.S.) 3Faculty of Experimental Science, Universidad Francisco de Vitoria, 28223 Madrid, Spain; [email protected] *Correspondence: [email protected] (B.O.-A.); [email protected] (A.J.M.-M.) Abstract: Phytoene (PT) and phytofluene (PTF), colorless carotenoids, have largely been ignored in food science studies, food technology, and nutrition. However, they are present in commonly consumed foods and may have health-promotion effects and possible uses as cosmetics. The goal of this study is to assess the most important food sources of PT and PTF and their dietary intakes in a representative sample of the adult Spanish population. A total of 62 food samples were analyzed ( 58 fruit and vegetables; seven items with different varieties/color) and carotenoid data of four foods (three fruits and one processed food) were compiled. PT concentration was higher than that of PTF in all the foods analyzed. The highest PT content was found in carrot, apricot, commercial tomato juice, and orange (7.3, 2.8, 2.0, and 1.1 mg/100 g, respectively). The highest PTF level was detected in carrots, commercial tomato sauce and canned tomato, apricot, and orange juice (1.7, 1.2, 1.0, 0.6, and 0.04 mg/100 g, respectively). The daily intakes of PT and PTF were 1.89 and 0.47 mg/person/day , respectively. The major contributors to the dietary intake of PT (98%) and PTF (73%) were: carrot, tomato, orange/orange juice, apricot, and watermelon. PT and PTF are mainly supplied by vegetables (81% and 69%, respectively). Considering the color of the edible part of the foods analyzed (fruit, vegetables, sauces, and beverages), the major contributor to the daily intake of PT and PTF (about 98%) were of red/orange color. Keywords: phytoene; phytofluene; food composition; carotenoid sources; carotenoid intake; Mediterranean diet 1. Introduction Carotenoids are ancient and widespread isoprenoids biosynthesized by photosynthetic organisms (cyanobacteria, algae, plants) as well as some fungi, bacteria, and a few known invertebrate animals [ 1 ]. Research on these compounds in the agro-food and health fields continues to grow due to their versatility and usefulness in developing healthy foods and other related products including functional foods, nutraceuticals, supplements, and novel foods [ 2 ]. Beyond their role as pigments and precursors of vitamin A (some of them), there is ample evidence that carotenoids can spark health-promoting biological actions contributing to a decrease in the risk of cancer, cardiovascular disease, and bone, skin, or eye disorders [ 3 , 4 ]. There is also increasing evidence that they can have positive effects on mental and metabolic health, during pregnancy and early life [ 5 , 6 ]. There is also a renewed interest in the use of carotenoids for cosmetics [7,8]. Phytoene (PT) is the precursor of the vast majority of carotenoids. The formation of phytoene from geranylgeranyl pyrophosphate (GGPP) is catalyzed by phytoene synthase, whereas phytoene desaturase introduces new double bonds to form phytofluene (PTF) and then other more unsaturated compounds [ 1 ]. Very high quantities of PT can be obtained by Nutrients 2021,13, 4436. https://doi.org/10.3390/nu13124436 https://www.mdpi.com/journal/nutrients Nutrients 2021,13, 4436 2 of 15 using bleaching compounds (for instance, norflurazom) that block carotenoid biosynthesis at the level of the desaturation of carotenes early in the pathway [ 9 ]. Very high levels are also found in fruit color mutants, such as Pinalate or Cara Cara oranges, resulting from abnormal carotenoid biosynthesis [ 10 , 11 ]. Moreover, as it can be inferred from the foods analyzed for this study, important amounts of PT and PTF can be present in plant foods also accumulating other carotenoids as a result of “normal” carotenoid biosynthesis. However, the colorless carotenoids PT and PTF have been largely ignored in food science and technology, and nutrition studies. This has changed in recent years as studies and reviews conclude that they are among the major carotenoids in many foods and could be involved in health-promoting actions in humans [ 12 – 17 ]. Although more studies are now reporting on the level of colorless carotenoids in foods are this information can be found in the latest comprehensive databases on food carotenoid content [ 18 – 20 ], carotenoid intakes are mostly unknown as they have only been assessed in a Luxembourg study [ 15 ]. The goal of this study is to report on the level of colorless carotenoids in commonly consumed foods in Spain, assess their daily intakes, and identify the major food contributors. 2. Materials and Methods 2.1. Fruits, Vegetables and Processed Food Samples Fruit and vegetables were acquired from supermarkets in Seville (Spain). A total of 62 samples were analyzed, 58 of which were fruits and vegetables, and four were ketchup, orange juice (from concentrate), tomato juice, and canned tomato. Different varieties/color of seven items were analyzed (guava, kiwi, lettuce, melon, peach, plum, pepper). Upon arrival to the laboratory, representative samples were chopped and freeze-dried. In the case of fruits, peel and the seeds were removed before processing. 2.2. Extraction and Rapid Resolution Liquid Chromatography (RRLC) Analysis of Carotenoids in Foods 50 mg samples (in triplicate) of homogenized freeze-dried powder were added 1 mL of extracting solvent (ethyl acetate/dichloromethane, 1:1 v/v) and then were vortexed. 700 µ L of water was then added and centrifuged at 18.000 × gfor 5 min. After recovering the colored fraction, an additional 500 µ L of dichloromethane was added and the mixture was vortexed and finally spun as described above. This operation was repeated until all color was gone. The pooled organic colored fractions were then evaporated to dryness in a vacuum concentrator (Eppendorf Concentrator Plus, Hamburg, Germany) and stored under N 2 at − 20 ◦ C until analysis. A saponification step was added in the case of samples with high carotenoid ester content. All carotenoid extracts were saponified except for those from carrot, sweet potato, watermelon, tomato and green fruits, and vegetables. To that end, extracts were redissolved in 500 µ L of dichloromethane and treated with 500 µ L of methanolic KOH (30%, w/v) overnight in a nitrogen atmosphere, dim light, and at room temperature. Lastly, the organic phase was washed with NaCl 5% until rinse water pH was neutral and then concentrated to dryness. The dry residue was re-dissolved in 50–100 µ L of acetonitrile before being injected into the RRLC system. Extracts were analyzed by RRLC with UV/VIS diode array detector according to a routine method [ 21 ]. They were identified by comparing their chromatographic and UV/vis spectroscopic characteristics with those of standards. PT and PTF were isolated from appropriate sources in accordance with standard procedures [ 22 ]. External calibration was used for quantification. The concentration of each standard was measured spectrophotometrically according to the conditions described in Britton et al. [ 23 ]. Full standard curves were constructed with five different concentrations for each carotenoid in triplicate. Subsequently, they were injected into the RRLC in triplicate. Both curves showed good linearity (R 2 = 0.999). The limit of detection ranged between 0.001 µ g for PTF and 0.002 µg for PT, while the limit of quantification ranged from 0.002 µg to 0.007 µ g for PTF and PT, respectively. Nutrients 2021,13, 4436 3 of 15 Extraction solvents (ethyl acetate and dichloromethane) were of analytical grade (VWR, Seattle, WA, USA). RRLC solvents, methanol (MeOH) and ethyl acetate, and acetonitrile were of RRLC grade and were acquired from Merck (Darmstadt, Germany). Water was purified in a NANOpure®DiamondTM system (Barnsted Inc., Dubuque, IO, USA). 2.3. Subjects A representative sample of the adult Spanish population (n= 3000), aged 18–64 years , took part in the last Spanish National Dietary Survey (Spanish acronym ENIDE) conducted by the Spanish Agency for Food Safety and Nutrition (AESAN) in 2009 and 2010, to determine dietary intake patterns in the adult Spanish population. The ENIDE study considered demographic characteristics (gender, age, geographical zone, size of family, and level of education), lifestyle and diet, and seasonal consumption, analyzing the same number of surveys in each of the four seasons of the year. Food consumption data were obtained from a 24-h dietary recall completed by each participant with the aid of trained interviewers and a three-day diet diary which together constituted a record of over 12,000 days of dietary consumption [ 24 ]. The ENIDE survey used this information to show the daily consumption of foods (n> 400 items), classified into 12 groups according to intake. Each food (raw) was expressed in grams or mL (in the case of liquids) per person per day, considering the overall population (consumers and non-consumers). The ENIDE survey gathered information on the consumption of mixed meals/recipes, recording both the amounts consumed by the participants and the way in which the meals were prepared. 2.4. Dietary Carotenoid Intake Assessment The mean of the food consumption data (g/person/day or ml/person/day) provided by the cross-section ENIDE survey [ 24 ] was used to assess the dietary intake of PT and PTF in the Spanish population. Data on the foods and amounts consumed were introduced into a specific software application for carotenoids [ 25 ] in which PT and PTF data content in 58 foods and 4 processed foods (orange juice from concentrate, ketchup, tomato: canned, juice) were analyzed in this study were included. PT and PTF concentrations in loquat, orange, pear, tomato sauce were obtained from the database of carotenoid content in Ibero-american foods [ 19 ]. PTF was not analyzed in loquat, orange, and pear, and PT was not analyzed in tomato sauce. The food groups included in the software are fruit, vegetables, oils and fats, snacks, nonalcoholic beverages, milk and dairy products, eggs and egg products, sauces, herbs, and spices. However, only data from foods belonging to the following groups were available: fruit, vegetables, sauces, non-alcoholic beverages. Foods considered in the calculations of the dietary intake of phytoene and phytofluene were (n= 66): (A) Fruits (n= 33, some of them of different varieties and colors): apple, apricot, avocado, banana, chestnut, flat peach, grapefruit, guava, guava (red), kaki, kiwi (green, yellow), lemon, loquat, mandarins, mango, melon (Cantaloupe), melon (white, yellow), nectarine, orange, orange juice (freshly squeeze), papaya, peach (gelo, igloo, red, yellow), pear, pineapple, plum (green, yellow), quince, watermelon. (B) Vegetables (n= 28, some of different varieties and colors): Artichoke, asparagus (green), beans (green), broccoli, cabbage, carrot, cauliflower, chard, corn, cucumber, eggplant, garlic (white), lamb’s lettuce, lettuce (“heart”, iceberg, romana), mushroom, peas, pepper (green, orange, red, yellow), potato, pumpkin, spinach, sweet potato, tomato, zucchini. (C) Juices and processed foods (n= 5): Ketchup, orange juice (from concentrate), tomato (canned, juice, sauce). The PT and PTF content in the foods consumed was multiplied by the amount of edible portion ingested (g/person/day) and this provided an estimate of the contribution of the food to the intake of these carotenoids. Intakes from individual foods were added up to yield individual and total carotenoid intake, as well as their relative contribution. A series of assumptions were made regarding the intake of the following foods and subsequent calculation of the carotenoid: artichoke (raw plus canned), lamb’s lettuce plus watercress/2, asparagus(greenandwhite)/2,kiwi(4/5greenand1/5yellow),lettuces( heart+ iceberg + romana/3 ), peach ( yellow + gelo + iglo + red/4 ),melon( white + yellow + galo/3 ),orangejuice( natural + concentrated ), Nutrients 2021,13, 4436 4 of 15 peppers ( yellow + orange + red + green/4 ), grapefruit (pink + yellow/2), carrot ( raw + boiled + canned ), prepared sauces (for example, tomato sauce, Bolognese sauce) were quantified considering the mean value of the concentrations of tomato paste and tomato puree reported in the food composition table [16]. The major food sources of PT and PTF in the diet of the Spanish population were thus determined. The contribution made by the group comprised of fruit and vegetables to the intake of these carotenoids is presented both for individual items and items grouped by color (white/yellow, green, red/orange). 3. Results 3.1. Phytoene and Phytofluene Concentration in Spanish Foods Table 1shows the concentration of PT and PTF in foods consumed by the Spanish population. Colorless carotenoids were detected in 21 of the 66 foods used in the dietary PT and PTF dietary assessment. Colorless carotenoids were found in traditional Mediterranean diet foods (such as pear, peaches, grapefruit, mandarin, nectarine, quince, watermelon, and tomato derivatives) and others that could be considered non-traditional or exotic (guavas, loquat, papaya) (Figure 1). The concentration of PT was higher than that of PTF in all the foods assessed except for tomato sauce in which no PT was detected. The highest PT contents were found in carrot, apricot, commercial tomato juice, and orange (7.3, 2.8, 2.0, and 1.1 mg/100 g, respectively). PT concentration in carrots was four times higher than that of PTF. The highest PTF levels were detected in carrot, commercial tomato sauce, and canned tomato, apricot, and orange juice (1.7, 1.2, 1.0, 0.6, and 0.04 mg/100 g, respectively). Nutrients 2021, 13, x FOR PEER REVIEW 7 of 15 Figure 1. Some sources of colorless carotenoids in Spanish dietary intake. 3.2. Daily Intakes of Phytoene and Phytofluene in Spain The contribution of each fruit and vegetable to the dietary intake of PT and PTF in the Spanish diet is shown in Table 1. The daily total intake of PT and PTF is 1889.2 µg/p/day and 470.4 µg/day respectively. However, intake may be slightly higher as PT and PTF data from four foods (tomato sauce, orange, pear, loquat) were taken from the Ibero-american carotenoid database [19], and these foods were not analyzed for PTF or PT. Five foods are major contributors to the dietary intake of PT and PTF: carrot, tomato, orange/orange juice, apricot, and watermelon, which account for 98% and 73% of the dietary intake of PT and PTF, respectively. Figure 2A,2B shows the major food contributors to the PT and PTF dietary intake. Figure 1. Some sources of colorless carotenoids in Spanish dietary intake. Nutrients 2021,13, 4436 5 of 15 Table 1. Phytoene and phytofluene content ( µ g/100 g fresh weight) (a) of fruits, vegetables and processed foods and daily intake ( µ g/person) and the consumption of fruits, vegetables, sauces and orange and tomato juices among the adult Spanish population. Name Spanish Name Scientific Name Edible Portion Content (µg)/100 g Fresh Weight (a) Food Intake (g/p/day) Dietary Intake (µg/day) Phytoene Phytofluene Phytoene Phytofluene Fruits Apple Manzana Malus domestica 80 41.4 0.0 0.0 Apricot Albaricoque Prunus armeniaca 93 2818 616 1.3 33.0 7.2 Avocado Aguacate Persea americana 72 1 0.0 0.0 Banana Plátano Musa ×paradisiaca 60 24.3 0.0 0.0 Chesnut Castaña Castanea sativa 82 0.2 0.0 0.0 Flat peach Paraguaya Prunus persica var. platycarpa 88 81 12 0.6 0.4 0.1 Grapefruit Pomelo Citrus ×paradisi 68 151 6 0.2 0.2 0.0 Guava Guayaba Psidium guajava 89 454 83 0.005 0.0 0.0 Guava (red) Guayaba (roja) Psidium guajava 89 187 32 0.005 0.0 0.0 Kaki Kaki Diospyros kaki 87 0.6 0.0 0.0 Kiwi (green) Kiwi (verde) Actinidia deliciosa 66 5.7 0.0 0.0 Kiwi (yellow) Kiwi (amarillo) Actinidia deliciosa 66 1.4 0.0 0.0 Lemon LimónCitrus ×limon 60 0.9 0.0 0.0 Loquat (b) Níspero Eriobotrya japonica 65 26 0.1 0.0 Mandarine Mandarina Citrus reticulata 73 60 51 9.8 4.3 3.7 Mango Mango Mangifera indica 68 0.4 0.0 0.0 Melon (cantaloupe) Melón galo Cucumis melo var. reticulatus 55 4.8 0.0 0.0 Melon (white) Melón piel de sapo Cucumis melo ‘Santa Claus’ 62 4.8 0.0 0.0 Melon (yellow) Melón (amarillo) Cucumis melo L. 60 4.8 0.0 0.0 Nectarine Nectarina Prunus persica var. nucipersica 89 29 6 0.04 0.0 0.0 Orange (b) Naranja Citrus ×sinensis 72 1065 34.6 265.6 Orange juice (freshly squeeze) Naranja (zumo natural) Citrus ×sinensis 100 122 40 11.9 14.5 4.8 Papaya Papaya Carica papaya 75 12 10 0.9 0.1 0.1 Peach gelo Melocotón gelo Prunus persica 88 3.8 0.0 0.0 Peach (igloo) Melocotón iglúPrunus persica 88 3.8 0.0 0.0 Peach (red) Melocotón (rojo) Prunus persica 69 99 14 3.8 2.6 0.4 Peach (yellow) Melocotón (amarillo) Prunus persica 69 26 2 3.8 0.7 0.1 Pear (b) Pera Pyrus communis 80 28.5 18.3 4.2 Nutrients 2021,13, 4436 6 of 15 Table 1. Cont. Name Spanish Name Scientific Name Edible Portion Content (µg)/100 g Fresh Weight (a) Food Intake (g/p/day) Dietary Intake (µg/day) Phytoene Phytofluene Phytoene Phytofluene Pineapple Piña Ananas comosus 57 6.7 0.0 0.0 Plum (green) Ciruela (verde) Prunus domestica subsp. domestica 85 1.0 0.0 0.0 Plum (yellow) Ciruela (amarilla) Prunus domestica subsp. domestica 92 1.0 0.0 0.0 Quince Membrillo Cydonia oblonga 61 116 44 0.4 0.2 0.1 Watermelon SandíaCitrullus lanatus 78 144 55 17.0 19.0 7.3 Subtotal 209.4 344.8 23.8 Vegetables Artichoke Alcachofa Cynara scolymus 47 2.6 0.0 0.0 Asparagus (green) Espárrago (verde) Asparagus officinalis 50 1.8 0.0 0.0 Beans (green) Judías (verdes) Phaseolus vulgaris var. vulgaris 93 8.3 0.0 0.0 Broccoli Brécol Brassica oleracea var. italica 97 0.1 0.0 0.0 Cabbage Col Brassica oleracea 2.3 0.0 0.0 Carrot Zanahoria Daucus carota 85 7264 1701 10.0 618.1 144.7 Cauliflower Coliflor Brassica oleracea var. botrytis 84 2.9 0.0 0.0 Chard Acelga Beta vulgaris var. Cicla 88 3.0 0.0 0.0 Corn MaízZea mays 100 1.8 0.0 0.0 Cucumber Pepino Cucumis sativus 70 4.6 0.0 0.0 Eggplant Berenjena Solanum melongena 85 3.4 0.0 0.0 Garlic (white) Ajo (blanco) Allium sativum 100 2.6 0.0 0.0 Lamb’s lettuce Canónigo Valerianella locusta 100 0.3 0.0 0.0 Lettuce (heart) Lechuga (cogollo) Lactiva longifolia 100 6.9 0.0 0.0 Lettuce (iceberg) Lechuga (iceberg) Lactuca sativa var. Capitata 88 6.9 0.0 0.0 Lettuce (romana) Lechuga (romana) Lactuca sativa 50 6.9 0.0 0.0 Mushroom ChampiñónAgaricus bisporus 80 5.6 0.0 0.0 Peas Guisante Pisum sativum 100 3.5 0.0 0.0 Pepper (green) Pimiento (verde) Capsicum annuum Group 95 3.4 0.0 0.0 Pepper (orange) Pimiento (naranja) Capsicum annuum Group 87 3.4 0.0 0.0 Pepper (red) Pimiento (rojo) Capsicum annuum Group 85 3.4 0.0 0.0 Pepper (yellow) Pimiento (amarillo) Capsicum annuum Group 81 3.4 0.0 0.0 Potato Patata Solanum tuberosum 74 68.4 0.0 0.0 Pumpkin Calabaza Cucurbita maxima 67 2.2 0.0 0.0 Spinach Espinaca Spinacia oleracea 76 4.4 0.0 0.0 Sweet potato Batata Ipomoea batatas 100 0.1 0.0 0.0 Nutrients 2021,13, 4436 7 of 15 Table 1. Cont. Name Spanish Name Scientific Name Edible Portion Content (µg)/100 g Fresh Weight (a) Food Intake (g/p/day) Dietary Intake (µg/day) Phytoene Phytofluene Phytoene Phytofluene Tomato Tomate Solanum lycopersicum 97 1697 330 55.6 915.2 178.0 Zucchini CalabacínCucurbita pepo 79 6.3 0.0 0.0 Subtotal 224.1 1533.3 322.7 Processed Foods Orange juice (from concentrate) Naranja (zumo a partir de concentrado) 100 0 0 11.9 0.0 0.0 Tomato canned Tomate enlatado 100 1878 999 0 0.0 0.0 Tomato juice Tomate en zumo 100 1994 884 0.4 7.8 3.4 Tomato sauce (b) Tomate en salsa 100 1165 10.3 119.6 Ketchup Kétchup 100 391 99 1.0 4.0 1.0 Subtotal 23.6 11.8 124.0 TOTAL 457.1 1889.9 470.5 (a) phytoene and phytofluene: empty boxes correspond to concentrations under detection limit. (b) data compiled from reference [19] and in these foods PT or PTF content were not analyzed. Nutrients 2021,13, 4436 8 of 15 3.2. Daily Intakes of Phytoene and Phytofluene in Spain The contribution of each fruit and vegetable to the dietary intake of PT and PTF in the Spanish diet is shown in Table 1. The daily total intake of PT and PTF is 1889.2 µ g/p/day and 470.4 µ g/day respectively. However, intake may be slightly higher as PT and PTF data from four foods (tomato sauce, orange, pear, loquat) were taken from the Ibero-american carotenoid database [ 19 ], and these foods were not analyzed for PTF or PT. Five foods are major contributors to the dietary intake of PT and PTF: carrot, tomato, orange/orange juice, apricot, and watermelon, which account for 98% and 73% of the dietary intake of PT and PTF, respectively. Figure 2A,B shows the major food contributors to the PT and PTF dietary intake. Nutrients 2021, 13, x FOR PEER REVIEW 8 of 15 (A) (B) Figure 2. Major contributors to the dietary intake of phytoene (A) and phytofluene (B). Table 2 shows the contribution of the different food groups to their daily intake. The main source of these colorless carotenoids is vegetables accounting for 81% of PT and 69% of PTF intake. Fruit and non-alcoholic beverages (orange juice and tomato juice) supply 352.8 and 27 µg/p/d of PT and PFT, respectively, accounting for 18.7% and 5.8% of the daily intake of PT and PTF, respectively. Figure 2. Major contributors to the dietary intake of phytoene (A) and phytofluene (B). Table 2shows the contribution of the different food groups to their daily intake. The main source of these colorless carotenoids is vegetables accounting for 81% of PT and 69% of PTF intake. Fruit and non-alcoholic beverages (orange juice and tomato juice) supply 352.8 and 27 µ g/p/d of PT and PFT, respectively, accounting for 18.7% and 5.8% of the daily intake of PT and PTF, respectively. Nutrients 2021,13, 4436 9 of 15 Table 2. Daily PT and PTF intake ( µ g/person/d) from the different food groups in the Spanish adult population. Food Group Phytoene Phytofluene Fruit 330.5 18.8 Vegetables 1533.3 322.7 Sauces 4.0 120.7 Non alcoholic beverages (orange juice, tomato juice) 22.3 8.2 Total 1890 470 Table 3shows the contribution of these foods (fruit, vegetables, sauces, and beverages) to the daily intake of PT and PTF grouped according to the color of their edible part. Red/orange colored foods are the major contributor to the daily intake of PT and PTF (about 98%), while white-yellowish foods account for 2% of the intake of the two carotenoids. Green foods do not supply these carotenoids. Table 3. Contribution of foods (fruit, vegetables, sauces, and beverages) grouped according to the color of their edible part to the daily intake (µg/person/d). Food Color Phytoene Phytofluene Red/orange 1852.0 463.0 Green 0.0 0.0 White-yellowish 37.9 7.4 4. Discussion 4.1. Phytoene and Phytofluene Concentrations in Spanish Foods PT and PTF were found in only about one-third of the foods (fruit, vegetables, sausages, and beverages) from those habitually consumed in the adult Spanish population [ 24 ]. The highest PT concentrations (>1 mg/100 g) were found in carrot, apricot, tomato (fresh, canned, juice), and orange (fresh) (7.3, 2.8, 2.0, and 1.1 mg/100 g, respectively). Carrot, tomato (canned, juice), and apricot are also the foods with the highest PTF concentrations (1.7, 1.0, 0.9, 0.6 mg/100 g, respectively). PT concentration was higher than that of PTF in all the foods analyzed, which is consistent with other studies [26]. PT and PTF concentrations in carrots are quite higher than those reported in other studies (1.3–1.8 and 0.6 mg/100 g for PT and PTF, respectively) [ 19 , 25 ]. In contrast, those found in apricot coincided with the values reported elsewhere [ 19 , 25 ]. Among Mediterranean fresh fruits, apricot appears to be the richest in colorless carotenoids, the main carotenoids found in commercially available varieties of this fruit [ 18 , 26 ]. Concentration in oranges is higher for PT in fresh orange and in the range of PT and PTF concentrations published in other studies (0.05–0.56 mg PT/100 g and 0.04–0.18 mg PTF/100 g) as tabulated in a review [ 26 ]. PT concentration in orange juice was nearly nine times lower than that found in the fresh fruit. This could be due to the fact that a large part of the pulp (where the carotenoids are found) is removed from the juice and the carotenoids break down during juicing as this process damages cell structures which could contribute to a carotenoid loss [ 26 ]. No PT or PTF was detected in orange juice made from concentrate. In this case, the thermal treatments this product is subjected to could contribute to carotenoid degradation [27]. The fact that highly variable concentrations of colorless carotenoids have been found in the literature for the same food is not surprising as the secondary metabolites content of fruits and vegetables is dependent on many factors, including the genotype, climate, agronomic practices as well as technological and/or culinary practices, among others [ 7 ]. As an example, tomatoes of the same variety grown under the same conditions have been known to exhibit differences in PT and PTF content of four-fold and six-fold, respectively [ 28 ]. As for differences across tomato cultivars, a recent study showed that PT levels ranged from